Ectodermal organ development is initiated by inductive tissue interactions. Developing teeth, epidermis, hair, and limbs are classic examples of these types of inductive processes. In mice, tooth development begins at embryonic day (E) 11.5 with thickening of the dental epithelium. The dental lamina undergoes further proliferation, subsequently developing into the tooth bud and germ. The tooth bud is formed by the invagination of the placode and the condensation of mesenchyme cells adjacent to the bud. At the cap stage (E14.5), dental epithelial cells differentiate into several cell types, such as inner dental epithelium (IDE), outer dental epithelium, stellate reticulum, and stratum intermedium. At the bell stage (E17.5), the IDE cells differentiate into enamel matrix-secreting ameloblasts, and the dental mesenchyme differentiates into dentin matrix-secreting odontoblasts. The goal of this project is to understand how tooth and craniofacial tissues develop and to define molecular defects underlying anomalies of these tissues. Mediator 1 acts as a coactivator for notch1 signaling and promotes transcription of the alkaline phosphatase gene: The Mediator is a multi-protein complex that functions as a transcriptional coactivator. The Mediator complex interacts with the core transcriptional machinery, including RNA polymerase II. Med1 is a subunit of the Mediator and was recently shown to be critical for hair formation, adipogenesis, and thyroid gland development. In collaboration with Dr. Yuko Oda at UCSF and Veterans Affairs Medical Center San Francisco, we previously showed that Med1 and Notch1 determine dental epithelial stem cell fate to the SI cell lineage and differentiation, as well as that it is required for enamel mineralization. Med1 deletion prevented Notch1-mediated differentiation of the SI cells resulting in decreased Alp1, which is essential for mineralization. However, it does not affect the ability of ameloblasts to produce enamel matrix proteins. More recently, using the dental epithelial SF2 cell line, we demonstrated that Med1 directly activates transcription of the Alpl gene through the stimulation of Notch1 signaling by forming a complex with activated Notch1/RBP-Jk on the Alpl promoter. These results suggest that Med1 is essential for enamel matrix mineralization by serving as a coactivator for Notch1 signaling regulating transcription of the Alpl gene. Epiprofin regulates enamel formation and tooth morphogenesis by controlling epithelial-mesenchymal interactions: The synchronization of cell proliferation and cytodifferentiation between dental epithelial and mesenchymal cells is required for the morphogenesis of teeth with correct functional shapes and optimum sizes. Epiprofin (Epfn), a transcription factor belonging to the Sp family, regulates dental epithelial cell proliferation and is essential for ameloblast and odontoblast differentiation. Epfn deficiency results in the lack of enamel and ironically the formation of extra teeth. We investigated the mechanism underlying the functions of Epfn in tooth development through the creation of transgenic mice expressing Epfn under the control of an epithelial cell-specific K5 promoter, (K5-Epfn). We found that these K5-Epfn mice developed abnormally shaped incisors and molars and formed fewer molars in the mandible. Ameloblasts differentiated ectopically, and enamel was formed on the lingual side of the K5-Epfn incisors. By contrast, ameloblasts and enamel were found only on the labial side in wild-type mice, as Follistatin (Fst) expressed on the labial side inhibits BMP4 signaling necessary for ameloblast differentiation. We showed that Epfn transfection into the dental epithelial cell line SF2 abrogated the inhibitory activity of Fst and promoted ameloblast differentiation of SF2 cells. We found that Epfn induced FGF9 in dental epithelial cells, and that this dental epithelial cell-derived FGF9 promoted dental mesenchymal cell proliferation via the FGF receptor 1c (FGFR1c). Taken together, these results suggest that Epfn regulates the balance between cell proliferation and cytodifferentiation in dental epithelial and mesenchymal cells during normal tooth development and morphogenesis. The novel basic-Helix-Loop-Helix transcription factor AmeloD regulates tooth morphogenesis: Basic-Helix-Loop-Helix (bHLH) transcription factors play important roles in various types of organogenesis. We identified the novel tooth-specific bHLH transcription factor AmeloD from a tooth germ cDNA library using the two-yeast hybrid system. AmeloD is expressed in the IDE but not in differentiated ameloblasts. To determine the role of AmeloD in tooth development, we created AmeloD KO mice. For mechanistic analysis, we used an AmeloD expression vector to examine the function of AmeloD in the dental epithelial cell line CLDE. We found that AmeloD KO mice developed enamel hypoplasia and small teeth because of inhibition of IDE cell division and migration and division through the increase of E-cadherin expression. Over-expression of AmeloD in CLDE cells suppressed endogenous E-cadherin expression and promoted cell division and migration. We also found that AmeloD contributed to the formation of multiple teeth in Epiprofin (Epfn) KO mice by strongly promoting dental epithelial cell motility. These findings demonstrate that AmeloD is an important factor that regulates tooth morphogenesis via the modulation of E-cadherin.
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